βig-h3 is an extracellular matrix protein whose expression is induced in various cell lines, including human melanoma cells, mammary ephithelial cells, keratinocytes, and lung fibroblasts, following signaling by active TGF-β (Skonier, J. et al., DNA Cell Biol. 13, 571, 1994). The βig-h3 gene was first isolated by differential hybridization screening of a cDNA library made from a human lung adenocarcinoma cell line that had been treated with TGF-β. βig-h3 gene encodes a 683-amino acid protein that is highly conserved between species. It contains an N-terminal secretory signal peptide and an Arg-Gly-Asp (RGD) motif at the C-terminus. The RGD motif is found in many extracellular matrix proteins modulating cell adhesion and serves as a ligand recognition sequence for several integrins (Stonier, J. et al., DNA Cell Biol., 11, 511, 1992).
According to several studies, βig-h3 is known to be involved in cell growth and proliferation, wound healing, and cell adhesion, although the underlying mechanisms for these functions are still unclear. However, βig-h3 seems to play an important role in the morphogenesis and interactions with cells and extracellular matrix proteins in various tissues.
Some evidence related to the role of βig-h3 in mediating cell attachment and detachment is provided by several studies. For example, purified βig-h3 protein is found to promote the attachment and spreading of skin fibroblasts while inhibiting the adhesion of A549, HeLa and Wi-38 cells in serum-free media. Particularly, βig-h3 is known to have inhibitory activity against tumor cell growth, and to affect colony formation and morphology. The inhibitory activity was demonstrated by the report in which transfection of βig-h3 expression plasmids into CHO (Chinese hamster ovary) cells led to marked decreases in cell proliferation and the ability of these cells to form tumors in nude mice. Further, a wound healing method was developed on the basis of the finding that application of a pharmaceutically effective amount of βig-h3 to wounds makes cells, especially fibroblasts, spread over and adhere to the wound site. Consequently, βig-h3, a cell adhesion molecule induced by TGF-β in various cell lines, plays a very important role in cell growth, cell differentiation, wound healing, morphogenesis and cell adhesion (Rawe, I. M. et al., Invest. Ophthalmol. Vis. Sci. 38, 893, 1997; Lebaron, R. G. et al., J. Invest. Dermatol. 104, 844, 1995).
βig-h3 contains four 140 amino acid repeats with internal homology, namely fas-1 domains. The internal repeat domains have highly conserved sequences found in secretory proteins or membrane proteins of various species, including mammals, insects, sea urchins, plants, yeasts, and bacteria. Proteins containing the conserved sequence are exemplified by periostin, fasciclin I, sea urchin HLC-2, algal-CAM and mycobacterium MPB70. The conserved domain in these proteins (hereinafter referred to as “fas-1”) consists of about 110 to 140 amino acids with two highly conserved branches, H1 and H2, of about 10 amino acids each (Kawamoto, T. et al., Biochem. Biophys. Acta. 1395, 288, 1998).
Four fas-1 domains are found in βig-h3, periostin, and fasciclin I, two fas-1 domains in HLC-2, and only one fas-1 domain in MPB70. Although the functions of the proteins are not elucidated clearly, some of them are known to act as cell adhesion molecules. For instance, βig-h3, periostin, and fasciclin 1 are reported to mediate the adhesion of fibroblasts, osteoblasts, and nerve cells, respectively. Also, it is disclosed that algal-CAM is a cell adhesion molecule present in embryos of the algae Volvox (LeBaron, R. G., et al., J. Invest. Dermatol. 104, 844, 1995; Horiuchi, K. et al., J. Bone Miner. Res. 14, 1239, 1999; Huber, O. et al., EMBO J. 13, 4212, 1994).
At first, it was believed that the cell attachment activity of βig-h3 would be mediated by the C-terminal RGD motif. However, some research results revealed that the RGD motif is not necessary for promoting the spreading of chondrocytes and that the mature soluble βig-h3 whose RGD motif is deleted by carboxyl-terminus processing is able to inhibit cell adhesion, leading to the conclusion that the RGD motif of βig-h3 is dispensable for mediating the cell attachment activity of βig-h3. In addition, it has been recently reported that βig-h3 promotes the spreading of fibroblasts via integrin α1β1 whereas the RGD motif of βig-h3 is not necessary for mediating the cell adhesion property of βig-h3. According to a recent report, βig-h3 binds specifically to integrin to enhance the cell adhesion and spreading of cells irrespective of RGD motif (Ohno, S. et al., Biochm. Biophys. Acta 1451, 196, 1999). Further, the conserved peptides H1 and H2 of βig-h3 were found to have no influence on βig-h3-mediated cell adhesion. These results, taken together, indicate that amino acids indispensable for the cell attachment activity of βig-h3 exist somewhere other than the H1 and H2 regions. A computer search based on homologies not only among the repeated fas-1 domains of βig-h3 but also among fas-1 domains of other proteins revealed that there are a few highly conserved amino acids in addition to H1 and H2 peptides, suggesting the possibility of the involvement of the conserved amino acid sequences in the cell attachment activity.
Of the domains of βig-h3, known to play an important role in cell adhesion, either of the 2nd or 4th domain is identified as a minimum domain essential for the cell adhesion of the molecule in accordance with the present invention. Based on these findings, recombinant proteins containing the essential functional domains are also identified as being effective for wound healing, in accordance with the present invention.
Recent research for wound healing has been subdivided into cell biology and molecular biology and the promotion of wound healing has had increasing applications in various clinical fields. However, cell biological and molecular biological mechanisms of wound healing still remain unclear. According to findings disclosed thus far, wound healing is a tissue response to trauma, leading to tissue repair through complex biological processes, including chemotaxis, cell differentiation and replication, matrix protein synthesis, angiogenesis, and wound reconstitution (Steed, D. L., et al., Clin. Plast. Surg. 25, 397, 1998).
Growth factors are representative materials that appear in the early stage of the wound healing process and control the subsequent wound healing process. Having strong influence over all stages of wound healing, growth factors act to control the growth, differentiation and metabolism of cells and reorganize the environs of the wound by their chemotactic properties which attract various cells types that are involved in inflammation and tissue repair, cellular proliferation, stimulating angiogenesis and the synthesis and degradation of the extracellular matrix. PDGF (platelet-derived growth factor) attracts fibroblasts to the wound and stimulates them to proliferate, and transforming growth factor-beta (TGF-β) causes them to make collagen. PDGF is chemotactic for most cells involved in wound healing, stimulates angiogenesis, remodeling and contraction, and activates wound healing cells (Mustoe, T. A. et al., J. Clin. Invest. 87, 694, 1991; Lepisto, J. et al., J. Surg. Res. 53, 596, 1992). EGF (epidermal growth factor) stimulates keratinocyte migration, angiogenesis and granulation tissue development and activates mitogenesis of keratinocyets and fibroblasts (Franklin, J. D. et al., Plast. Recsonst. Surg. 64, 766, 1979; Buckly, A. et al., Proc. Natl. Acad. Sci. USA, 82, 7340, 1985). bFGF (basic fibroblast growth factor) stimulates angiogenesis, epithelialization, and collagenous fiber deposition, and associates with heparin in various forms to perform relevant functions (Tsuboi, R. et al., J. Exp. Med. 172, 245, 1990; Kinsnorth, A. N. et al., Br. J. Surg. 77, 409, 1990). IGF (insulin-like growth factor) enhances cell differentiation. VEGF (vascular endothelial growth factor) increases vasopermeability and promotes endothelial mitogenesis.
Of the growth factors and cytokines involved in wound healing, TGF-β is the most representative. Existing in three forms (TGF-β1, TGF-β2 and TGF-β3) in mammals, the cytokine plays important roles in the growth and differentiation of various cells and has various complex functions, including control of cell growth, regulation of immune responses, stimulation of osteogenesis, induction of cartilage specific macromolecules, and promotion of wound healing (Bennett, N. T. et al., Am. J. Surg. 165, 728, 1993). Appearing in the ephithelium during wound healing, TGF-β is believed to stimulate the expression of integrin within keratinocytes during re-epithelialization. In recent research into TGF-β expression, it was revealed that TGF-β3 mRNA is expressed in the epithelia of normal skin and acute and chronic wounds, while TGF-β1 mRNA is not expressed in normal skin and chronic wounds, but expressed in the epithelial layer regenerated from acute wounds, and nowhere is expressed TGF-β2 mRNA (Schmid, P. et al., J. Pathol. 171, 191, 1993). Based on the effects, even though their mechanisms are not firmly established, TGF-β is expected to play a major role in re-epithelialization.
Expression of βig-h3 is up-regulated by TGF-β, suggesting that βig-h3 is involved in the mediation of some signals of TGF-β. CHO (Chinese hamster ovary) cells transformed with βig-h3 expression plasmids are reported to show decreased tumorigenic ability (Skonier, J. et al., DNA Cell Biol. 13, 571, 1994). In contrast, βig-h3 expression is down-regulated in dexamethasone-treated stem cells, some tumor cells and the fibroblasts cultured from the skin lesion sites afflicted with localized hyperostosis of melorheostosis. βig-h3 is also reported to serve as a negative regulator of osteogenesis (Genini, M. et al., Int. J. Cancer 66, 571, 1996; Schenker, T. et al., Exp. Cell. Res. 239, 161, 1998; Kim, J. et al., J. Cell Biochem. 77, 169, 2000). In addition to these functions, βig-h3, known as a cell adhesion molecule, promotes the adhesion and spreading of fibroblasts in the dermis. According to studies into the distribution of βig-h3 in eye tissues, it is reported that the adhesion molecule is expressed in corneal epithelia of normal adults, intracorneal fetal stromal cells, and the endothelial and stromal cells in the process of wound healing. In addition, βig-h3 is expressed in the juxtaglomerular apparatus and proximal tubules of the kidneys, and its expression is increased in diabetes mellitus. Further, it is found in subendothelial smooth muscles of the coronary arteries of normal persons, and its amount is increased in the endometria of blood vessels in the case of arteriosclerosis. However, the expression of βig-h3 in normal dermal tissues and dermal wounds has not yet been firmly established (Klintworth, G. K. et al., Am. J. Pathol. 152, 743, 1998; Munier, F. L. et al., Nature Genetics 15, 247, 1997; Streeten B. W. et al., Arch. Ophthalmol. Vis. Sci. 38, 893, 1997). As mentioned above, the distribution and expression of βig-h3 in normal human tissues remains unclear. Particularly, there are no reports regarding expression patterns of βig-h3 in dermal wounds. However, some research groups have reported that βig-h3 functions to promote the adhesion and spreading of dermal fibroblasts, so that it is expected to make a contribution to the promotion of wound healing.